Hyang-Bok Lee

Isolation and anti‐inflammatory effect of astragalin synthesized by enzymatic hydrolysis of tea seed extract

BACKGROUND The application of tea seed extract (TSE) has been widely investigated because of its biological activities. In this paper, two flavonol triglycosides in TSE-camelliaside A (CamA) and camelliaside B (CamB)-were subjected to hydrolysis in the presence of two commercial enzyme complexes (Pectinex™ series): Smash and Mash. RESULTS Smash hydrolyzed only the xylosyl moiety of CamB, and the main product was kaempferol diglycoside (nicotiflorin, NF). On the other hand, Mash induced the hydrolysis of both CamA and CamB, and kaempferol monoglycoside (astragalin, AS) was found to be a main product. Pure AS with > 96% purity was prepared by enzymatic hydrolysis of TSE using Mash, and the chemical structure of AS was confirmed by (1)H- and (13)C-nuclear magnetic resonance analyses. The prepared pure AS showed anti-inflammatory activities by significantly inhibiting cellular nitrite oxide (IC(50) = 363 µg mL(-1)), prostaglandin E(2) (IC(50) = 134 µg mL(-1)) and interleukin-6 production (IC(50) = 289 µg mL(-1)) by lipopolysaccharide -stimulated RAW 264.7 cells. CONCLUSION It was concluded that pure AS can be prepared by enzymatic partial hydrolysis of TSE and employed as an anti-inflammatory material. This is the first study to address the preparation of pure AS from natural sources.

Isolation and characterization of nicotiflorin obtained by enzymatic hydrolysis of two precursors in tea seed extract.

Two flavonol triglycosides, camelliaside A (CamA) and camelliaside B (CamB), of tea seed extract (TSE) were subjected to enzymatic hydrolysis. Among five kinds of glycosidases investigated, beta-galactosidase (Gal) induced selective hydrolysis of CamA. On the other hand, pectinase (Pec) and cellulase (Cel) induced hydrolysis of CamB. For Gal and Pec, only kaempferol diglycoside (nicotiflorin, NF) was produced; on the other hand, significant amounts of kaempferol monoglycoside (astragalin, AS) and kaempferol (KR) were also detected for Cel. The combination of the use of Gal and Pec in the enzymatic hydrolysis of TSE afforded NF with high specificity. Crude NF with 22% purity was recovered from the enzymatic reaction mixture by extraction with organic solvent, and pure NF with >95% purity was obtained by crystallized in water. The chemical structure of NF was confirmed by (1)H and (13)C NMR analyses.

Homology modelling and virtual screening of P-protein in a quest for novel antimelanogenic agent and in vitro assessments.

An adequate knowledge on molecular mechanism of melanogenesis provides an opportunity to find the novel molecular targets for the discovery and development of new cosmetics. Among various genes, the OCA2 is being essential for proper melanin synthesis, and mutation or deletion of this gene leads to oculocutaneous albinism type 2. Thus, for this study, the product of this gene, that is P‐protein, was targeted in quest for novel inhibitors as antimelanogenic agents. Based on pattern search of amino acid sequence and homology analysis, the protein structure was modelled. The role of this protein has been predicted as a tyrosine transporter of melanosomes. Thus, the molecular library was generated on the basis of tyrosine transporter inhibitor. Based on the dock score, 20 molecules have been considered as putative inhibitors for P‐protein. Among these compounds, five molecules (compound #1, #4, #8, #13 and #17) were found to be quite effective as antimelanogenic without having any toxicity. Further investigations to establish the mechanism of action, the indirect methods such as tyrosinase assay, analysis for eumelanin and pheomelanins and investigation of mRNA levels were being carried out. The results from the studies offered a new lead in antimelanogenic therapy and may be very useful for further optimization work in developing them as novel depigmenting agents.

Unrevealing the role of P-protein on melanosome biology and structure, using siRNA-mediated down regulation of OCA2

Abstract The pink-eyed dilution protein (P-protein) plays a critical role in melanin synthesis in melanocytes and retinal pigment epithelium cells. Mutation in this protein may cause complete or partial albinism. Role of the P-protein ranges in melanin synthesis to maturation and trafficking of the melanosomes. The aim of the present study was to evaluate the effect of P-protein inhibition on melanosome biology by comparing the shape, size, count, and types of melanosomes in melan-a melanocytes. The cells were extensively examined by the transmission electron microscopy. The P-protein inhibition was carried by P-protein-siRNA transfection to melan-a melanocytes, B16F10 mouse melanoma, and melan-p1 cells. Measurement of melanin contents, cellular tyrosinase, and different tyrosinase related proteins were also determined to investigate the effect of P-protein siRNA transfection on melanocytes. Results suggested that the inhibition of P-protein can significantly change the melanosomal morphology, types and their respective numbers, and provided a novel strategy for the control of melanin synthesis.

A small molecule inhibitor of Mitf-E-box DNA binding and its depigmenting effect in melan-a cells

Background  Microphthalmia associated transcription factor (Mitf) is a key regulatory transcriptional factor of pigmentation‐related genes including tyrosinase. Inhibition of tyrosinase transcription by blocking the binding of Mitf with its promoter E‐box DNA can control the pigmentation. However, no such chemicals were reported so far.

Anti-melanogenic effect of Prunus davidiana extract in melan-a melanocyte through regulation of OCA-2, TRP-1 and tyrosinase

Prunus spp. and locally available plants (used as folkloric medicine) were screened to find a novel and natural anti-melanogenic agent. Based on p-protein promoter reporter assay (PPRA) the candidate plants were screened in the quest for p-protein inhibitor. Expression profiling of key proteins revealed the molecular mechanism of the melanin inhibition as well as TEM analysis revealed melanosome structure. The screened plant extract through PPRA showed significant down regulation of p-protein, which led to melanin inhibition. Another key melanosomal protein like tyrosinase and TRP-1 was also found to be down-regulated. However, TRP-2 was not affected. TEM analysis of treated cells also revealed that the stage IV melanosomes were lowered in number compared to control. The present study shows the plants used in this study possess good anti-melanogenic properties. However, the P. davidiana has the highest anti-melanogenic property among screened plant extracts.